Oligomerization of expanded-polyglutamine domain fluorescent fusion proteins in cultured mammalian cells

Six inherited neurologic diseases, including Huntington's disease, result from the expansion of a CAG domain of the disease genes to produce a domain of more than 40 glutamines in the expressed protein. The mechanism by which expansion of this polyglutamine domain causes disease is unknown. Recent studies demonstrated oligomerization of polyglutamine-domain proteins in mammalian neurons. To study oligomerization of polyglutamine proteins and to identify heterologous protein interactions, varying length polyglutamine-green fluorescent protein fusion proteins were expressed in cultured COS-7 cells. The 19- and 35-glutamine fusion proteins (non-pathologic length) distributed diffusely throughout the cytoplasm. In contrast, 56- and 80-glutamine fusion proteins (pathologic length) formed fibrillar arrays resembling those previously observed in neurons in Huntington's disease and in a transgenic mouse model. These aggregates were intranuclear and intracytoplasmic. Intracytoplasmic aggregates were surrounded by collapsed intermediate filaments. The intermediate filament protein vimentin co-immunoisolated with expanded polyglutamine fusion proteins. This cellular model will expedite investigations into oligomerization of polyglutamine proteins and their interactions with other proteins.     

Crosslinking of apolipoprotein E by products of lipid peroxidation

Apolipoprotein E (APOE) genotype and advancing aging are interacting ri sk factors in the expression of late onset and sporadic Alzheimer's Disease (AD). We tested the hypothesis that 2 products of lipid peroxidation, malondialdehyde (MDA) and 4 hydroxy-2-nonenal (HNE), covalently modify APOE and alter its metabolism. In vitro, both HNE and MDA crosslinked purified APOE3 and APOE4. HNE was a more potent crosslinker than MDA, and purified APO3 was more susceptible to crosslinking by HNE than was purified APOE4. In P19 neuroglial cultures, oxidative stress with lipid peroxidation led to increased intracellular accumulation of anti-HNE and anti-APOE immunoreactive proteins of approximately 50 kDa. Intercellular accumulation of the 50 kDa APOE-immunoreactive protein (APOE-50) was not prevented by cyclohexamide, suggesting formation by post-translational mechanisms. In CSF, a 50 kDa APOE-immunoreactive protein co-migrated with proteins most immunoreactive for HNE and MDA adducts, containing NaB3H4-reducible bonds. These proteins were in CSF from adult subjects (with or without dementia), and in AD patients homozygous for APOE3 or APOE4 alleles. These data suggest that HNE covalently crosslinks APOE in P19 neuroglial cultures to form a 50 kDa protein, and that similar modifications of APOE appear to occur in vivo.     

Pattern of elevation of urine catecholamines in intracerebral haemorrhage

Autonomic nervous system dysfunction is a common complication of severe intracranial disease. The aim of this study was to reveal the autonomic changes in patients suffering from acute intracerebral haemorrhage (ICH). 25 patients with spontaneous ICH within 24 hours of onset of symptoms were included. All patients were treated with standardised medical management and the meta- and normetanephrines were detected by high performance liquid chromatography (HPLC) in 24-hour urine every day. The mean level of normetanephrine (709 +/- 579 micrograms/day) and metanephrine (244 +/- 161 mg/day) were significantly elevated in comparison with a control group, p < or = 0.01. The norepinephrine elevation was of greater diagnostic and prognostic importance. Maximum urinary catecholamine metabolite levels occurred between day 3 to 10 after the bleeding. Normetanephrines correlated with the prognosis and the complications of ICH: intraventricular involvement resulted in significantly elevated normetanephrine levels (896 +/- 520 micrograms/day versus 311 +/- 78 micrograms/day) p < or = 0.01. Patients with a great volume of haematoma developed severe autonomic dysregulation (normetanephrines 1114 +/- 493 micrograms/day), whereas patients with smaller haematoma did not (339 +/- 125 micrograms/day) p < or = 0.0001; patients with bad outcome (1014 +/- 620 mg/day) had higher levels of normetanephrines than those with a good prognosis (322 +/- 110 micrograms/day) p < or = 0.001. A close relationship to elevated intracranial pressure was established. This study demonstrated the feasibility of detecting autonomic nervous system dysfunction in neurological intensive care patients by means of examination of the metabolites of the catecholamines in the urine.(ABSTRACT TRUNCATED AT 250 WORDS)     

GAP-43 augmentation of G protein-mediated signal transduction is regulated by both phosphorylation and palmitoylation

The neuronal protein GAP-43 is concentrated at the growth cone membrane, where it is thought to amplify the signal transduction process. As a model for its neuronal effects, GAP-43 protein injection into Xenopus laevis oocytes strongly augments the calcium-sensitive chloride current evoked by the G protein-coupled receptor stimulation. We have now examined a series of GAP-43 mutants in this system and determined those regions of GAP-43 required for this increase in current flux. As expected, palmitoylation inhibits signal amplification in oocytes by blocking G protein activation. Unexpectedly, a second domain of GAP-43 (residues 35-50) containing a protein kinase C phosphorylation site at residue 41 is also necessary for augmentation of G protein-coupled signals in oocytes. This region is not required for activation of isolated Go but is necessary for GAP-43 binding to isolated calmodulin and to isolated protein kinase C. Substitution of Asp for Ser41 inactivates GAP-43 as a signal facilitator in oocytes. This mutation blocks GAP-43 binding to both protein kinase C and calmodulin. Thus, GAP-43 regulates an oocyte signaling cascade via coordinated, simultaneous G protein activation and interaction with either calmodulin or protein kinase C.     

Novel large apolipoprotein E-containing lipoproteins of density 1.006-1.060 g/ml in human cerebrospinal fluid

Although the critical role of apolipoprotein E (apoE) allelic variation in Alzheimer's disease and in the outcome of CNS injury is now recognized, the functions of apoE in the CNS remain obscure, particularly with regard to lipid metabolism. We used density gradient ultracentrifugation to identify apoE-containing lipoproteins in human CSF. CSF apoE lipoproteins, previously identified only in the 1.063-1.21 g/ml density range, were also demonstrated in the 1.006-1.060 g/ml density range. Plasma lipoproteins in this density range include low-density lipoprotein and high-density lipoprotein (HDL) subfraction 1 (HDL1). The novel CSF apoE lipoproteins are designated HDL1. No immunoreactive apolipoprotein A-I (apo A-I) or B could be identified in the CSF HDL1 fractions. Large lipoproteins 18.3 +/- 6.6 nm in diameter (mean +/- SD) in the HDL1 density range were demonstrated by electron microscopy. Following fast protein liquid chromatography of CSF at physiologic ionic strength, apoE was demonstrated in particles of average size greater than particles containing apoA-I. The largest lipoproteins separated by this technique contained apoE without apoA-I. Thus, the presence of large apoE-containing lipoproteins was confirmed without ultracentrifugation. Interconversion between the more abundant smaller apoE-HDL subfractions 2 and 3 and the novel larger apoE-HDL1 is postulated to mediate a role in cholesterol redistribution in brain.     

Somatostatin-like immunoreactivity, its molecular forms and monoaminergic metabolites in aged and demented patients with Parkinson's disease--effect of L-Dopa

There is some evidence that Parkinson's disease (PD) seems to be a heterogenous and generalized brain disorder reflecting a degeneration of multiple neuronal networks, including somatostatinergic neurons. Somatostatin-like immunoreactivity (SLI) and its molecular forms, high molecular weight form (HMV-SST), somatostatin-14 (SST-14), somatostatin-25/28 (SST-25/28) and Des-ala-somatostatin (Des-ala-SST), as well as homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) were estimated using HPLC and radioimmunoassay in the cerebrospinal fluid (CSF) of 35 aged parkinsonian patients with different stages of intellectual deterioration. The influence of L-dopa-treatment on these neurochemical parameters was evaluated. Without a correlation with dementia scores (p = 0.11), SLI was significantly reduced in PD in comparison to the control group (p < 0.05). The reduction was related to the progression of the disease. Correlations between SLI, HVA and 5-HIAA indicate a heterogenous brain disorder in PD with alterations of several transmitter systems and functions. Complex qualitative and quantitative changes in the molecular pattern of SLI are compatible with a dysregulated synthesis and/or posttranslational processing. L-dopa-treatment was associated with a significant increase of HVA (p < 0.05) and HMV-SST (p < 0.05) and a slight, but insignificant increase of SLI (p = 0.11).     

Nitride Laser Diodes With Nonepitaxial Cladding Layers

Semiconductor laser diodes (LDs) typically use lattice-matched epitaxial layers as waveguide cladding materials. We describe a new LD architecture in which the upper cladding layer is replaced with an evaporated or sputtered nonepitaxial material. Designs and results are presented for 415-nm InGaN LDs that use indium tin oxide, silver, or a silver–palladium–copper alloy as the cladding material, chosen because the material exhibits requisite optical and electrical properties. The nonepitaxial cladding layer offers several advantages, such as eliminating the need to expose vulnerable InGaN active layers to the high temperatures required for growing conventional p-AlGaN cladding layers subsequent to the active layer growth.      

Long-Time Stability of Ni-Ti-Shape Memory Alloys for Automotive Safety Systems

In automotive a lot of electromagnetically, pyrotechnically or mechanically driven actuators are integrated to run comfort systems and to control safety systems in modern passenger cars. Using shape memory alloys (SMA) the existing systems could be simplified, performing the same function through new mechanisms with reduced size, weight, and costs. A drawback for the use of SMA in safety systems is the lack of materials knowledge concerning the durability of the switching function (long-time stability of the shape memory effect). Pedestrian safety systems play a significant role to reduce injuries and fatal casualties caused by accidents. One automotive safety system for pedestrian protection is the bonnet lifting system. Based on such an application, this article gives an introduction to existing bonnet lifting systems for pedestrian protection, describes the use of quick changing shape memory actuators and the results of the study concerning the long-time stability of the tested NiTi-wires. These wires were trained, exposed up to 4 years at elevated temperatures (up to 140 °C) and tested regarding their phase change temperatures, times, and strokes. For example, it was found that A _P-temperature is shifted toward higher temperatures with longer exposing periods and higher temperatures. However, in the functional testing plant a delay in the switching time could not be detected. This article gives some answers concerning the long-time stability of NiTi-wires that were missing till now. With this knowledge, the number of future automotive applications using SMA can be increased. It can be concluded, that the use of quick changing shape memory actuators in safety systems could simplify the mechanism, reduce maintenance and manufacturing costs and should be insertable also for other automotive applications.     

A Comprehensive Investigation On The Application Of Ozone In Cooling Water Systems –– Correlation of Bench–Top,Pilot Scale and Field Application Data

The first comprehensive study – bench–top laboratory investigations, pilot scale testing, and critical monitoring and evaluation of field applications – addressing the effects of ozone as a stand–alone cooling water treatment program is presented. The study also represents the first critical comparison of ozone–treated systems with non–treated systems. Excellent corrosion control can be attained in ozone–treated cooling water systems. However, the corrosion rates are completely dominated by the water chemistry of the system and have no dependence on the presence of ozone at typical use levels. Good control of fouling can also be attained. However, as was the case with corrosion control, deposition on the heatexchange surfaces is not determined by the presence of ozone, but by several factors that traditionally influence fouling in a system. The strong biocidal properties of ozone resulted in excellent microbiological control in all Pilot Cooling Tower (PCT) investigations and in both case studies. Excellent agreement was observed among all stages of testing.